PROJECT SUMMARY/ABSTRACT A sustained inflammatory environment eventually leads to irreversible damage to dental pulp tissue. Traditionally, a severely inflamed dental pulp is removed and replaced with inert materials via root canal therapy (RCT). However, devitalized teeth are brittle, and may be more susceptible to structural failure. Therefore, it is desirable to preserve the vital pulp by developing more effective therapeutic interventions to resolve pulpitis and to create a microenvironment that is conducive to healing and regeneration. Thus, targeting multiple factors that concurrently impact the transition from the inflammatory to the regenerative phase are likely to prove more effective than the application of a single factor-driven treatment. Mesenchymal stem cells (MSCs) stand out as a good candidate for generating multiple factors to drive the transition from the inflammatory to the regeneration phase. MSCs have been widely reported to possess properties for multifunctional therapeutic applications and have been shown to be a rich source of factors that promote tissue repair and resolve of inflammatory. However, substantial concerns regarding their safety and efficacy during the pre-clinical and clinical applications have dramatically restricted their use as a direct regenerative tool. Cell-derived decellularized extracellular matrices (DMs) therefore represent an alternative approach to confer bioactivity from such cells to direct cell fate and response when applied to recipient cells or tissues. Guided by this rationale, we propose studies that will test our central hypothesis that DMs, captured in biomimetic self-assembling multidomain peptide hydrogels (MDPs) when delivered within a controlled cultural environment will direct the resolution of inflammation and regeneration. We have generated MDPs that are biocompatible, injectable and self-assembling nanofibrillar scaffolds and shown that these 3D tissue constructs with multiple aligned cell layers can be simply generated within MDPs. Moreover, in controlled culture conditions, MSCs cultured within MDPs produce bioactive factors that are sequestered within the nanofibrillar environment. The DMs captured within MDPs (DM-MDPs) have the ability to retain sufficient bioactivity to direct cell behaviors even after they decellularization. Based upon these compelling data, we would like to test our central hypothesis by determining whether DM-MDPs can be used for controlling inflammation and sustaining cell growth in an ex vivo mandible organ culture model system (Aim 1). Aim 2 will apply the optimized DM-MDPs in a rodent dental pulp injury model to evaluate whether these conditioned scaffolds will recapitulate their therapeutic effects in an in vivo system.